Within the broad framework of regenerative medicine, musculoskeletal regeneration poses particular challenges. While advances in stem cell biology and tissue engineering have brought us closer to the goal of being able to produce physiologically competent tissues, including cartilage, bone, muscle and tendons, the function of the musculoskeletal system requires that these tissues be organized and integrated with one another and with the nervous system to function successfully. Thus, while the ability to enhance repair of local muscle or skeletal damage is improving all the time and will likely continue to do so at an accelerating rate in the future, the capacity to regenerate a functional musculoskeletal system after more catastrophic loss remains a distant dream. A major reason that there has been so little progress in this critical area has been the lack of a tractable animal model for addressing it. While zebrafish can regenerate their fins and are well suited to molecular and genetic analyses, the portion of the fin capable of regenerating (the fin rays) contains neither endochondral bone nor muscle and tendons. Hence, the only class of vertebrates capable of regenerating entire musculoskeletal structures is the urodele amphibians, the salamanders and their relatives. However, research on amphibian limb regeneration has remained a relative backwater due to the lack of experimental tools. Utilizing the axolotl, a salamander that is highly suited for use in a laboratory setting, this proposal is to develop methods and transgenic animals that will allow the problem of limb regeneration to be addressed on a modern genetic level. This will include creating a system for tissue-specific manipulation of gene activity (such that the roles of genes in specific regenerating tissues can be assessed), for conditionally regulating gene expression (such that development of the limb can occur normally and gene activation or knock-down can be initiated only during specific stages of the regenerative process). Moreover, methods will be established for rapidly altering gene expression at a high efficiency with viral vectors in addition to definitive gene manipulation in transgenic animals. Establishing such a genetic system will be transformative for future work towards regenerating an integrative musculoskeletal system in higher organisms. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a genetic system for studying the regeneration of integrated musculoskeletal systems such as is found in the limb. Transgenic approaches in axolotls will be utilized to develop a means of activating and knocking down gene function with tissue-specificity and temporal control.